Studies on the mechanisms and regulation of clathrin-mediated endocytosis (CME) and actin force generation during CME, and their critical importance to cell function in both budding yeast and mammalian cells, are proposed. Actin functions in countless processes including cell motility, organelle transport, adhesion, contractility, cell shape, cell polarity, and maintenance of membrane tension and cell mechanical rigidity. Significant gaps exist in knowledge of actin mechanisms and assembly regulation. Two key questions concerning actin regulation and function will be addressed in studies of budding yeast: (1) How does the cell cycle regulate actin cable assembly? (2) How do type 1 myosin and the Arp2/3 complex work together to create forces that generate membrane curvature? For the former studies, recent observation that fimbrin phosphorylation by Clb2/Cdk1 is crucial for cell cycle regulation of actin assembly will be leveraged to develop a mechanistic understanding of how actin assembly is regulated in the cell cycle. For the latter studies, in- depth biochemical, biophysical, genetic, and cell biological approaches will be combined to determine how type 1 myosins contribute to force production by Arp2/3-nucleated actin networks during CME. CME is responsible for uptake of molecules from a cell's environment through the permeability barrier of the plasma membrane, and therefore, is crucial for determining how cells respond to their surroundings. Many proteins and lipids that mediate CME have been identified, and their functions determined biochemically and in cells. Live cell imaging of fluorescently labeled CME proteins has revealed the intricate recruitment timing and order for some 60 CME proteins. However, how cargo capture is coordinated with vesicle formation, how correct protein recruitment order and timing are achieved, which events and molecules play critical roles in the pathway, and how forces curve the membrane and drive vesicle scission, are not fully understood. The following key questions will be addressed in budding yeast and mammalian cells: How are CME site initiation and maturation regulated? What activities are essential for CME vesicle formation? Does a checkpoint monitor CME? What biophysical principles govern CME? What are actin's endocytic functions and how are they regulated? How do chemical and physical parameters affect CME dynamics and efficiency? How does CME change during cellular differentiation? Mammalian cell studies will be conducted on over 80 stable tissue culture and stem cell lines generated using genome editing to express CME proteins as fluorescent protein fusions at native, endogenous levels. Effects of cell differentiation on CME dynamics and efficiency will be conducted in the genome-edited stem cells. Because CME proteins are highly conserved in structure and function, principles learned from studies of yeast and mammals will each complement and inform the other and provide a comprehensive mechanistic understanding that neither alone could generate.